Soybean cultivar 4442112

ABSTRACT

A soybean cultivar designated 4442112 is disclosed. The invention relates to the seeds of soybean cultivar 4442112, to the plants of soybean 4442112, to plant parts of soybean cultivar 4442112 and to methods for producing a soybean plant produced by crossing soybean cultivar 4442112 with itself or with another soybean variety. The invention also relates to methods for producing a soybean plant containing in its genetic material one or more transgenes and to the transgenic soybean plants and plant parts produced by those methods. This invention also relates to soybean cultivars or breeding cultivars and plant parts derived from soybean variety 4442112, to methods for producing other soybean cultivars, lines or plant parts derived from soybean cultivar 4442112 and to the soybean plants, varieties, and their parts derived from use of those methods. The invention further relates to hybrid soybean seeds, plants and plant parts produced by crossing the cultivar 4442112 with another soybean cultivar.

BACKGROUND OF THE INVENTION

The present invention relates to a new and distinctive soybean cultivar,designated 4442112. All publications cited in this application areherein incorporated by reference.

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possess the traits to meetthe program goals. The goal is to combine in a single variety animproved combination of desirable traits from the parental germplasm.These important traits may include higher seed yield, resistance todiseases and insects, better stems and roots, tolerance to drought andheat, and better agronomic quality.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, and recurrent selection.

The complexity of inheritance influences choice of the breeding method.Backcross breeding is used to transfer one or a few favorable genes fora highly heritable trait into a desirable cultivar. This approach hasbeen used extensively for breeding disease-resistant cultivars. Variousrecurrent selection techniques are used to improve quantitativelyinherited traits controlled by numerous genes. The use of recurrentselection in self-pollinating crops depends on the ease of pollination,the frequency of successful hybrids from each pollination and the numberof hybrid offspring from each successful cross.

Each breeding program should include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulcultivars produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested and compared toappropriate standards in environments representative of the commercialtarget area(s) for three or more years. The best lines are candidatesfor new commercial cultivars; those still deficient in a few traits maybe used as parents to produce new populations for further selection.

These processes, which lead to the final step of marketing anddistribution, usually take from eight to 12 years from the time thefirst cross is made. Therefore, development of new cultivars is atime-consuming process that requires precise forward planning, efficientuse of resources, and a minimum of changes in direction.

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

The goal of soybean plant breeding is to develop new, unique andsuperior soybean cultivars and hybrids. The breeder initially selectsand crosses two or more parental lines, followed by repeated selfing andselection, producing many new genetic combinations. The breeder cantheoretically generate billions of different genetic combinations viacrossing, selfing and mutations. The breeder has no direct control atthe cellular level. Therefore, two breeders will never develop the sameline, or even very similar lines, having the same soybean traits.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions and further selections arethen made during and at the end of the growing season. The cultivarsthat are developed are unpredictable because the breeder's selectionoccurs in unique environments with no control at the DNA level (usingconventional breeding procedures), and with millions of differentpossible genetic combinations being generated. A breeder of ordinaryskill in the art cannot predict the final resulting lines he develops,except possibly in a very gross and general fashion. The same breedercannot produce the same cultivar twice by using the same originalparents and the same selection techniques. This unpredictability resultsin the expenditure of large amounts of research monies to developsuperior new soybean cultivars.

The development of new soybean cultivars requires the development andselection of soybean varieties, the crossing of these varieties andselection of superior hybrid crosses. The hybrid seed is produced bymanual crosses between selected male-fertile parents or by using malesterility systems. These hybrids are selected for certain single genetraits such as pod color, flower color, pubescence color or herbicideresistance which indicate that the seed is truly a hybrid. Additionaldata on parental lines, as well as the phenotype of the hybrid,influence the breeder's decision whether to continue with the specifichybrid cross.

Pedigree breeding and recurrent selection breeding methods are used todevelop cultivars from breeding populations. Breeding programs combinedesirable traits from two or more cultivars or various broad-basedsources into breeding pools from which cultivars are developed byselfing and selection of desired phenotypes. The new cultivars areevaluated to determine which have commercial potential.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops. Two parents that possess favorable,complementary traits are crossed to produce an F₁. An F₂ population isproduced by selfing one or several F₁s. Selection of the bestindividuals may begin in the F₂ population; then, beginning in the F₃,the best individuals in the best families are selected. Replicatedtesting of families can begin in the F₄ generation to improve theeffectiveness of selection for traits with low heritability. At anadvanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new cultivars.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified, or created,by intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line which is the recurrent parent. The source of the trait tobe transferred is called the donor parent. After the initial cross,individuals possessing the phenotype of the donor parent are selectedand repeatedly crossed (backcrossed) to the recurrent parent. Theresulting plant is expected to have the attributes of the recurrentparent (e.g., cultivar) and the desirable trait transferred from thedonor parent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

In a multiple-seed procedure, soybean breeders commonly harvest one ormore pods from each plant in a population and thresh them together toform a bulk. Part of the bulk is used to plant the next generation andpart is put in reserve. The procedure has been referred to as modifiedsingle-seed descent or the pod-bulk technique.

The multiple-seed procedure has been used to save labor at harvest. Itis considerably faster to thresh pods with a machine than to remove oneseed from each by hand for the single-seed procedure. The multiple-seedprocedure also makes it possible to plant the same number of seeds of apopulation each generation of inbreeding. Enough seeds are harvested tomake up for those plants that did not germinate or produce seed.

In addition to phenotypic observations, the genotype of a plant can alsobe examined. There are many laboratory-based techniques available forthe analysis, comparison and characterization of plant genotype; amongthese are Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Amplified Fragment Length polymorphisms (AFLPs), Simple Sequence Repeats(SSRs—which are also referred to as Microsatellites), and SingleNucleotide Polymorphisms (SNPs).

Isozyme Electrophoresis and RFLPs have been widely used to determinegenetic composition. Shoemaker and Olsen, (Molecular Linkage Map ofSoybean (Glycine max L. Merr.) p 6.131–6.138 in S. J. O'Brien (ed)Genetic Maps: Locus Maps of Complex Genomes, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., (1993)) developed amolecular genetic linkage map that consisted of 25 linkage groups withabout 365 RFLP, 11 RAPD and three classical markers and four isozymeloci. See also, Shoemaker, R. C., RFLP Map of Soybean, p 299–309, inPhillips, R. L. and Vasil, I. K. (eds.) DNA-Based Markers in Plants,Kluwer Academic Press, Dordrecht, the Netherlands (1994).

SSR technology is currently the most efficient and practical markertechnology; more marker loci can be routinely used and more alleles permarker locus can be found using SSRs in comparison to RFLPs. Forexample, Diwan and Cregan described a highly polymorphic microsatellitelocus in soybean with as many as 26 alleles. (Diwan, N. and Cregan, P.B., Theor. Appl. Genet. 95:22–225, 1997.) SNPs may also be used toidentify the unique genetic composition of the invention and progenyvarieties retaining that unique genetic composition. Various molecularmarker techniques may be used in combination to enhance overallresolution.

Molecular markers, which includes markers identified through the use oftechniques such as Isozyme Electrophoresis, RFLPs, RAPDs, AP-PCR, DAF,SCARs, AFLPs, SSRs, and SNPs, may be used in plant breeding. One use ofmolecular markers is Quantitative Trait Loci (QTL) mapping. QTL mappingis the use of markers which are known to be closely linked to allelesthat have measurable effects on a quantitative trait. Selection in thebreeding process is based upon the accumulation of markers linked to thepositive effecting alleles and/or the elimination of the markers linkedto the negative effecting alleles from the plant's genome.

Molecular markers can also be used during the breeding process for theselection of qualitative traits. For example, markers closely linked toalleles or markers containing sequences within the actual alleles ofinterest can be used to select plants that contain the alleles ofinterest during a backcrossing breeding program. For example, molecularmarkers are used in soybean breeding for selection of the trait ofresistance to soybean cyst nematode, see U.S. Pat. No. 6,162,967. Themarkers can also be used to select toward the genome of the recurrentparent and against the markers of the donor parent. Using this procedurecan attempt to minimize the amount of genome from the donor parent thatremains in the selected plants. It can also be used to reduce the numberof crosses back to the recurrent parent needed in a backcrossingprogram. The use of molecular markers in the selection process is oftencalled Genetic Marker Enhanced Selection. Molecular markers may also beused to identify and exclude certain sources of germplasm as parentalvarieties or ancestors of a plant by providing a means of trackinggenetic profiles through crosses as discussed more fully hereinafter.

Mutation breeding is another method of introducing new traits intosoybean varieties. Mutations that occur spontaneously or areartificially induced can be useful sources of variability for a plantbreeder. The goal of artificial mutagenesis is to increase the rate ofmutation for a desired characteristic. Mutation rates can be increasedby many different means including temperature, long-term seed storage,tissue culture conditions, radiation (such as X-rays, Gamma rays,neutrons, Beta radiation, or ultraviolet radiation), chemical mutagens(such as base analogues like 5-bromo-uracil), antibiotics, alkylatingagents (such as sulfur mustards, nitrogen mustards, epoxides,ethyleneamines, sulfates, sulfonates, sulfones, or lactones), azide,hydroxylamine, nitrous acid or acridines. Once a desired trait isobserved through mutagenesis the trait may then be incorporated intoexisting germplasm by traditional breeding techniques. Details ofmutation breeding can be found in “Principles of Cultivar Development”by Fehr, Macmillan Publishing Company, 1993.

The production of double haploids can also be used for the developmentof homozygous varieties in a breeding program. Double haploids areproduced by the doubling of a set of chromosomes from a heterozygousplant to produce a completely homozygous individual. For example, seeWan et al., Theor. Appl. Genet., 77:889–892, 1989.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Allard, 1960; Simmonds, 1979; Sneep et al., 1979; Fehr,1987).

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, the grower, processor and consumer for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new cultivar should take into consideration research and developmentcosts as well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

Soybean, Glycine max (L), is an important and valuable field crop. Thus,a continuing goal of soybean plant breeders is to develop stable, highyielding soybean cultivars that are agronomically sound. The reasons forthis goal are obviously to maximize the amount of grain produced on theland used and to supply food for both animals and humans. To accomplishthis goal, the soybean breeder must select and develop soybean plantsthat have traits that result in superior cultivars.

SUMMARY OF THE INVENTION

According to the invention, there is provided a novel soybean cultivardesignated 4442112. This invention thus relates to the seeds of soybeancultivar 4442112, to the plants of soybean 4442112 and to methods forproducing a soybean plant produced by crossing the soybean 4442112 withitself or another soybean line, and the creation of variants bymutagenesis or transformation of soybean 4442112.

Thus, any such methods using the soybean cultivar 4442112 are part ofthis invention: selfing, backcrosses, hybrid production, crosses topopulations, and the like. All plants produced using soybean cultivar4442112 as a parent are within the scope of this invention.Advantageously, the soybean cultivar could be used in crosses withother, different, soybean plants to produce first generation (F₁)soybean hybrid seeds and plants with superior characteristics.

In another aspect, the present invention provides for single or multiplegene converted plants of 4442112. The transferred gene(s) may preferablybe a dominant or recessive allele. Preferably, the transferred gene(s)will confer such traits as herbicide resistance, insect resistance,resistance for bacterial, fungal, or viral disease, male fertility, malesterility, enhanced nutritional quality, and industrial usage. The genemay be a naturally occurring soybean gene or a transgene introducedthrough genetic engineering techniques.

In another aspect, the present invention provides regenerable cells foruse in tissue culture of soybean plant 4442112. The tissue culture willpreferably be capable of regenerating plants having the physiologicaland morphological characteristics of the foregoing soybean plant, and ofregenerating plants having substantially the same genotype as theforegoing soybean plant. Preferably, the regenerable cells in suchtissue cultures will be embryos, protoplasts, meristematic cells,callus, pollen, leaves, anthers, roots, root tips, flowers, seeds, podsor stems. Still further, the present invention provides soybean plantsregenerated from the tissue cultures of the invention.

Definitions

In the description and tables that follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Allele. Allele is any of one or more alternative forms of a gene, all ofwhich alleles relate to one trait or characteristic. In a diploid cellor organism, the two alleles of a given gene occupy corresponding locion a pair of homologous chromosomes.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotypes of the F₁hybrid.

Brown Stem Rot. This is a visual disease score from 1 to 9 comparing allgenotypes in a given test. The score is based on leaf symptoms ofyellowing and necrosis caused by brown stem rot. Visual scores rangefrom a score of 9, which indicates no symptoms, to a score of 1 whichindicates severe symptoms of leaf yellowing and necrosis.

Cotyledon. A cotyledon is a type of seed leaf. The cotyledon containsthe food storage tissues of the seed.

Embryo. The embryo is the small plant contained within a mature seed.

Emergence. This score indicates the ability of the seed to emerge whenplanted 3″ deep in sand at a controlled temperature of 25° C. The numberof plants that emerge each day are counted. Based on this data, eachgenotype is given a 1 to 9 score based on its rate of emergence andpercent of emergence. A score of 9 indicates an excellent rate andpercent of emergence, an intermediate score of 5 indicates averageratings and a 1 score indicates a very poor rate and percent ofemergence.

Hilum. This refers to the scar left on the seed that marks the placewhere the seed was attached to the pod prior to the seed beingharvested.

Hypocotyl. A hypocotyl is the portion of an embryo or seedling betweenthe cotyledons and the root. Therefore, it can be considered atransition zone between shoot and root.

Iron-Deficiency Chlorosis. Plants are scored 1 to 9 based on visualobservations. A score of 9 means no stunting of the plants or yellowingof the leaves and a score of 1 indicates the plants are dead or dyingcaused by iron-deficiency chlorosis, a score of 5 means plants haveintermediate health with some leaf yellowing.

Lodging Resistance. Lodging is rated on a scale of 1 to 9. A score of 9indicates erect plants. A score of 5 indicates plants are leaning at a45° angle in relation to the ground and a score of 1 indicates plantsare lying on the ground.

Maturity Date. Plants are considered mature when 95% of the pods havereached their mature color. The number of days are calculated eitherfrom August 31 or from the planting date.

Maturity Group. This refers to an agreed-on industry division of groupsof varieties based on zones in which they are adapted, primarilyaccording to day length or latitude. They consist of very long daylength varieties (Groups 000, 00, 0), and extend to very short daylength varieties (Groups VII, VIII, IX, X).

Relative Maturity (RM). The term relative maturity is a numerical valuethat is assigned to a soybean variety based on comparisons with thematurity values of other varieties. The number preceding the decimalpoint in the RM refers to the maturity group. The number following thedecimal point refers to the relative earliness or lateness within eachmaturity group. For example, a 3.0 is an early group III variety, whilea 3.9 is a late group III variety.

Oil or oil percent. Soybean seeds contain a considerable amount of oil.Oil is measured by NIR spectrophotometry, and is reported on an as ispercentage basis.

Oleic Acid Percent. Oleic acid is one of the five most abundant fattyacids in soybean seeds. It is measured by gas chromatography and isreported as a percent of the total oil content.

Palmitic Acid Percent. Palmitic acid is one of the five most abundantfatty acids in soybean seeds. It is measured by gas chromatography andis reported as a percent of the total oil content.

Phytophthora Tolerance. Tolerance to Phytophthora root rot is rated on ascale of 1 to 9, with a score of 9 being the best or highest toleranceranging down to a score of 1 which indicates the plants have notolerance to Phytophthora.

Phenotypic Score. The Phenotypic Score is a visual rating of generalappearance of the variety. All visual traits are considered in the scoreincluding healthiness, standability, appearance and freedom of disease.Ratings are scored from 1 being poor to 9 being excellent.

Plant Height. Plant height is taken from the top of soil to the top nodeof the plant and is measured in centimeters.

Pod. This refers to the fruit of a soybean plant. It consists of thehull or shell (pericarp) and the soybean seeds.

Protein Percent. Soybean seeds contain a considerable amount of protein.Protein is generally measured by NIR spectrophotometry, and is reportedon an as is percentage basis.

Pubescence. This refers to a covering of very fine hairs closelyarranged on the leaves, stems and pods of the soybean plant.

Quantitative Trait Loci (QTL). Quantitative trait loci (QTL) refer togenetic loci that control to some degree numerically representabletraits that are usually continuously distributed.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

Seed Protein Peroxidase Activity. Seed protein peroxidase activity isdefined as a chemical taxonomic technique to separate cultivars based onthe presence or absence of the peroxidase enzyme in the seed coat. Thereare two types of soybean cultivars, those having high peroxidaseactivity (dark red color) and those having low peroxidase activity (nocolor).

Seed Yield (Bushels/Acre). The yield in bushels/acre is the actual yieldof the grain at harvest.

Seeds per Pound. Soybean seeds vary in seed size, therefore, the numberof seeds required to make up one pound also varies. This affects thepounds of seed required to plant a given area, and can also impact enduses.

Shattering. The amount of pod dehiscence prior to harvest. Poddehiscence involves seeds falling from the pods to the soil. This is avisual score from 1 to 9 comparing all genotypes within a given test. Ascore of 9 means pods have not opened and no seeds have fallen out. Ascore of 5 indicates approximately 50% of the pods have opened, withseeds falling to the ground and a score of 1 indicates 100% of the podsare opened.

Single Gene Converted (Conversion). Single gene converted (conversion)plant refers to plants which are developed by a plant breeding techniquecalled backcrossing wherein essentially all of the desired morphologicaland physiological characteristics of a variety are recovered in additionto the single gene transferred into the variety via the backcrossingtechnique or via genetic engineering.

DETAILED DESCRIPTION OF THE INVENTION

4442112 is an early maturity group 0 variety with resistance to ROUNDUPherbicide conferring tolerance to glyphosate herbicides. 4442112 alsohas the Rps 1 a gene for resistance to Phytophthora root rot. 4442112has very high yield potential when compared to lines of similar maturityand has excellent agronomic characteristics including lodgingresistance.

Some of the selection criteria used for various generations include:seed yield, lodging resistance, emergence, disease tolerance, maturity,late season plant intactness, plant height and shattering resistance.

The cultivar has shown uniformity and stability, as described in thefollowing variety description information. It has been self-pollinated asufficient number of generations with careful attention to uniformity ofplant type. The line has been increased with continued observation foruniformity.

Soybean cultivar 4442112 has the following morphologic and othercharacteristics (based primarily on data collected at Adel, Iowa).

Table 1 Variety Description Information

Seed Coat Color (Mature Seed): Yellow

Seed Coat Luster (Mature Hand Shelled Seed): Dull

Cotyledon Color (Mature Seed): Yellow

Leaflet Shape: Ovate

Growth Habit: Indeterminate

Flower Color: Purple

Hilum Color (Mature Seed): Yellow

Plant Pubescence Color: Gray

Pod Color: Brown

Maturity Group: 0

Relative Maturity: 0.3

Plant Lodging Score: 8

Plant Height (cm): 71

Seed Size (# Seeds/lb.): 2730

Seed Content:

-   -   % Protein: 34.4    -   % Oil: 19.0

Physiological Responses: ROUNDUP READY Herbicide Resistant

Disease Resistance: Phytophthora Root Rot—Rps 1 a

This invention is also directed to methods for producing a soybean plantby crossing a first parent soybean plant with a second parent soybeanplant, wherein the first or second soybean plant is the soybean plantfrom the line 4442112. Further, both first and second parent soybeanplants may be from the cultivar 4442112. Therefore, any methods usingthe cultivar 4442112 are part of this invention: selfing, backcrosses,hybrid breeding, and crosses to populations. Any plants produced usingcultivar 4442112 as a parent are within the scope of this invention.

Useful methods include but are not limited to expression vectorsintroduced into plant tissues using a direct gene transfer method suchas microprojectile-mediated delivery, DNA injection, electroporation andthe like. More preferably expression vectors are introduced into planttissues using microprojectile-mediated delivery with a biolistic deviceor using Agrobacterium-mediated transformation. Transformant plantsobtained with the protoplasm of the invention are intended to be withinthe scope of this invention.

Soybean cultivar 4442112 is similar to 18020800. While similar to108020800, there are numerous differences including: 4442112 has thegene for ROUNDUP resistance while 18020800 does not contain this gene.

FURTHER EMBODIMENTS OF THE INVENTION

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes”. Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed variety or line.

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector comprises DNAcomprising a gene under control of, or operatively linked to, aregulatory element (for example, a promoter). The expression vector maycontain one or more such operably linked gene/regulatory elementcombinations. The vector(s) may be in the form of a plasmid, and can beused alone or in combination with other plasmids, to provide transformedsoybean plants, using transformation methods as described below toincorporate transgenes into the genetic material of the soybeanplant(s).

Expression Vectors for Soybean Transformation: Marker Genes—Expressionvectors include at least one genetic marker, operably linked to aregulatory element (a promoter, for example) that allows transformedcells containing the marker to be either recovered by negativeselection, i.e., inhibiting growth of cells that do not contain theselectable marker gene, or by positive selection, i.e., screening forthe product encoded by the genetic marker. Many commonly used selectablemarker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or an herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene which, when under thecontrol of plant regulatory signals, confers resistance to kanamycin.Fraley et al., Proc. Natl. Acad. Sci. U.S.A., 80:4803 (1983). Anothercommonly used selectable marker gene is the hygromycinphosphotransferase gene which confers resistance to the antibiotichygromycin. Vanden Elzen et al., Plant Mol. Biol., 5:299 (1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase and aminoglycoside-3′-adenyltransferase, the bleomycin resistance determinant. Hayford et al., PlantPhysiol. 86:1216 (1988), Jones et al., Mol. Gen. Genet., 210:86 (1987),Svab et al., Plant Mol. Biol. 14:197 (1990) Hille et al., Plant Mol.Biol. 7:171 (1986). Other selectable marker genes confer resistance toherbicides such as glyphosate, glufosinate or bromoxynil. Comai et al.,Nature 317:741–744 (1985), Gordon-Kamm et al., Plant Cell 2:603–618(1990) and Stalker et al., Science 242:419–423 (1988).

Selectable marker genes for plant transformation not of bacterial origininclude, for example, mouse dihydrofolate reductase, plant5-enolpyruvylshikimate-3-phosphate synthase and plant acetolactatesynthase. Eichholtz et al., Somatic Cell Mol. Genet. 13:67 (1987), Shahet al., Science 233:478 (1986), Charest et al., Plant Cell Rep. 8:643(1990).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include β-glucuronidase (GUS),β-galactosidase, luciferase and chloramphenicol acetyltransferase.Jefferson, R. A., Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al., EMBOJ. 8:343 (1989), Koncz et al., Proc. Natl. Acad. Sci. USA 84:131 (1987),DeBlock et al., EMBO J. 3:1681 (1984).

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissue are available. Molecular Probes publication2908, IMAGENE GREEN, p. 1–4 (1993) and Naleway et al., J. Cell Biol.115:151a (1991). However, these in vivo methods for visualizing GUSactivity have not proven useful for recovery of transformed cellsbecause of low sensitivity, high fluorescent backgrounds and limitationsassociated with the use of luciferase genes as selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells. Chalfie et al., Science 263:802 (1994). GFP and mutants of GFPmay be used as screenable markers.

Promoters—Genes included in expression vectors must be driven by anucleotide sequence comprising a regulatory element, for example, apromoter. Several types of promoters are well known in thetransformation arts as are other regulatory elements that can be usedalone or in combination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred”.Promoters that initiate transcription only in a certain tissue arereferred to as “tissue-specific”. A “cell-type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter that is active under mostenvironmental conditions.

Inducible Promoters—An inducible promoter is operably linked to a genefor expression in soybean. Optionally, the inducible promoter isoperably linked to a nucleotide sequence encoding a signal sequencewhich is operably linked to a gene for expression in soybean. With aninducible promoter the rate of transcription increases in response to aninducing agent.

Any inducible promoter can be used in the instant invention. See Ward etal., Plant Mol. Biol. 22:361–366 (1993). Exemplary inducible promotersinclude, but are not limited to, that from the ACEI system whichresponds to copper (Mett et al., PNAS 90:4567–4571 (1993)); In2 genefrom maize which responds to benzenesulfonamide herbicide safeners(Hershey et al., Mol. Gen Genetics 227:229–237 (1991) and Gatz et al.,Mol. Gen. Genetics 243:32–38 (1994)) or Tet repressor from Tn10 (Gatz etal., Mol. Gen. Genetics 227:229–237 (1991)). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone. Schena etal., Proc. Natl. Acad. Sci. USA 88:0421 (1991).

Constitutive Promoters—A constitutive promoter is operably linked to agene for expression in soybean or the constitutive promoter is operablylinked to a nucleotide sequence encoding a signal sequence which isoperably linked to a gene for expression in soybean.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell et al., Nature 313:810–812 (1985)) and the promoters from suchgenes as rice actin (McElroy et al., Plant Cell 2: 163–171 (1990));ubiquitin (Christensen et al., Plant Mol. Biol. 12:619–632 (1989) andChristensen et al., Plant Mol. Biol. 18:675–689 (1992)); PEMU (Last etal., Theor. Appl. Genet. 81:581–588 (1991)); MAS (Velten et al., EMBO J.3:2723–2730 (1984)) and maize H3 histone (Lepetit et al., Mol. Gen.Genetics 231:276–285 (1992) and Atanassova et al., Plant Journal 2 (3):291–300 (1992)).

The ALS promoter, Xba1/Ncol fragment 5′ to the Brassica napus ALS3structural gene (or a nucleotide sequence similarity to said Xba1/Ncolfragment), represents a particularly useful constitutive promoter. SeePCT application WO96/30530.

Tissue-specific or Tissue-preferred Promoters—A tissue-specific promoteris operably linked to a gene for expression in soybean. Optionally, thetissue-specific promoter is operably linked to a nucleotide sequenceencoding a signal sequence which is operably linked to a gene forexpression in soybean. Plants transformed with a gene of interestoperably linked to a tissue-specific promoter produce the proteinproduct of the transgene exclusively, or preferentially, in a specifictissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferredpromoter—such as that from the phaseolin gene (Murai et al., Science23:476–482 (1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci.USA 82:3320–3324 (1985)); a leaf-specific and light-induced promotersuch as that from cab or rubisco (Simpson et al., EMBO J.4(11):2723–2729 (1985) and Timko et al., Nature 318:579–582 (1985)); ananther-specific promoter such as that from LAT52 (Twell et al., Mol.Gen. Genetics 217:240–245 (1989)); a pollen-specific promoter such asthat from Zm13 (Guerrero et al., Mol. Gen. Genetics 244:161–168 (1993))or a microspore-preferred promoter such as that from apg (Twell et al.,Sex. Plant Reprod. 6:217–224 (1993)).

Signal Sequences for Targeting Proteins to SubcellularCompartments—Transport of protein produced by transgenes to asubcellular compartment such as the chloroplast, vacuole, peroxisome,glyoxysome, cell wall or mitochondrion or for secretion into theapoplast, is accomplished by means of operably linking the nucleotidesequence encoding a signal sequence to the 5′ and/or 3′ region of a geneencoding the protein of interest. Targeting sequences at the 5′ and/or3′ end of the structural gene may determine, during protein synthesisand processing, where the encoded protein is ultimatelycompartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample, Becker et al., Plant Mol. Biol. 20:49 (1992); Close, P. S.,Master's Thesis, Iowa State University (1993); Knox, C., et al., PlantMol. Biol. 9:3–17 (1987); Lerner et al., Plant Physiol. 91:124–129(1989); Frontes et al., Plant Cell 3:483–496 (1991); Matsuoka et al.,Proc. Natl. Acad. Sci. 88:834 (1991); Gould et al., J. Cell. Biol.108:1657 (1989); Creissen et al., Plant J. 2:129 (1991); Kalderon, etal., Cell 39:499–509 (1984); Steifel, et al., Plant Cell 2:785–793(1990).

Foreign Protein Genes and Agronomic Genes—With transgenic plantsaccording to the present invention, a foreign protein can be produced incommercial quantities. Thus, techniques for the selection andpropagation of transformed plants, which are well understood in the art,yield a plurality of transgenic plants which are harvested in aconventional manner, and a foreign protein then can be extracted from atissue of interest or from total biomass. Protein extraction from plantbiomass can be accomplished by known methods which are discussed, forexample, by Heney and Orr, Anal. Biochem. 114:92–6 (1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is a soybean plant. In anotherpreferred embodiment, the biomass of interest is seed. For therelatively small number of transgenic plants that show higher levels ofexpression, a genetic map can be generated, primarily via conventionalRFLP, PCR and SSR analysis, which identifies the approximate chromosomallocation of the integrated DNA molecule. For exemplary methodologies inthis regard, see Glick and Thompson, Methods in Plant Molecular Biologyand Biotechnology CRC Press, Boca Raton 269:284 (1993). Map informationconcerning chromosomal location is useful for proprietary protection ofa subject transgenic plant. If unauthorized propagation is undertakenand crosses made with other germplasm, the map of the integration regioncan be compared to similar maps for suspect plants, to determine if thelatter have a common parentage with the subject plant. Map comparisonswould involve hybridizations, RFLP, PCR, SSR and sequencing, all ofwhich are conventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

1. Genes that Confer Resistance to Pests or Disease and that Encode:

A. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance gene(s) to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266:789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262:1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinoset al. Cell 78:1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae).

B. A gene conferring resistance to a pest, such as soybean cystnematode. See e.g., PCT Application WO96/30517; PCT ApplicationWO93/19181.

C. A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser et al., Gene48:109 (1986), who disclose the cloning and nucleotide sequence of a Btδ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxin genes canbe purchased from American Type Culture Collection, Manassas, Va., forexample, under ATCC Accession Nos. 40098, 67136, 31995 and 31998.

D. A lectin. See, for example, Van Damme et al., Plant Molec. Biol.24:25 (1994), who disclose the nucleotide sequences of several Cliviaminiata mannose-binding lectin genes.

E. A vitamin-binding protein such as avidin. See PCT applicationUS93/06487 which teaches the use of avidin and avidin homologues aslarvicides against insect pests.

F. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem.262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor), Huub et al., Plant Molec. Biol. 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor 1), Sumitani etal., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus α-amylase inhibitor) and U.S. Pat. No.5,494,813 (Hepher and Atkinson, issued Feb. 27, 1996).

G. An insect-specific hormone or pheromone such as an ecdysteroid orjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

H. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem. 269:9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor), and Pratt etal., Biochem. Biophys. Res. Comm. 163:1243 (1989) (an allostatin isidentified in Diploptera puntata). See also U.S. Pat. No. 5,266,317 toTomalski et al., who disclose genes encoding insect-specific, paralyticneurotoxins.

I. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang et al., Gene 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

J. An enzyme responsible for a hyperaccumulation of a monoterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

K. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 (Scott et al.), which discloses the nucleotidesequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hornworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene.

L. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104:1467 (1994), who provide the nucleotide sequenceof a maize calmodulin cDNA clone.

M. A hydrophobic moment peptide. See PCT application WO95/16776, whichdiscloses peptide derivatives of Tachyplesin which inhibit fungal plantpathogens, and PCT application WO95/18855 which teaches syntheticantimicrobial peptides that confer disease resistance.

N. A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure of Jaynes et al., Plant Sci 89:43 (1993), ofheterologous expression of a cecropin-β lytic peptide analog to rendertransgenic tobacco plants resistant to Pseudomonas solanacearum.

O. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. Rev. Phytopathol.28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virusand tobacco mosaic virus.

P. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Cf.Taylor et al., Abstract #497, Seventh Int'l Symposium on MolecularPlant-Microbe Interactions (Edinburgh, Scotland) (1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

Q. A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

R. A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo-α-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase. See Lamb et al., Bio/Technology10:1436 (1992). The cloning and characterization of a gene which encodesa bean endopolygalacturonase-inhibiting protein is described by Toubartet al., Plant J. 2:367 (1992).

S. A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bio/Technology 10:305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

T. Genes involved in the Systemic Acquired Resistance (SAR) Responseand/or the pathogenesis related genes. Briggs, S. Current Biology, 5(2)(1995).

U. Antifungal genes. See Cornelissen and Melchers, Plant Physiol.,101:709–712 (1993); Parijs et al., Planta 183:258–264 (1991) andBushnell et al., Can. J. of Plant Path. 20(2):137–149 (1998).

V. Genes that confer resistance to Phytophthora root rot, such as theRps 1, Rps 1-a, Rps 1-b, Rps 1-c, Rps 1-d, Rps 1-e, Rps 1-k, Rps 2, Rps3-a, Rps 3-b, Rps 3-c, Rps 4, Rps 5, Rps 6, Rps 7 and other Rps genes.See, for example, Shoemaker et al., Phytophthora Root Rot ResistanceGene Mapping in Soybean, Plant Genome IV Conference, San Diego, Calif.(1995).

2. Genes that Confer Resistance to an Herbicide, for Example:

A. An herbicide that inhibits the growing point or meristem, such as animidazalinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449(1990), respectively.

B. Glyphosate (resistance conferred by mutant5-enolpyruvlshikimate-3-phosphate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus PAT bar genes), and pyridinoxy or phenoxy proprionic acidsand cyclohexones (ACCase inhibitor-encoding genes). See, for example,U.S. Pat. No. 4,940,835 to Shah, et al., which discloses the nucleotidesequence of a form of EPSP which can confer glyphosate resistance. A DNAmolecule encoding a mutant aroA gene can be obtained under ATCCaccession number 39256, and the nucleotide sequence of the mutant geneis disclosed in U.S. Pat. No. 4,769,061 to Comai. European patentapplication No. 0 333 033 to Kumada et al., and U.S. Pat. No. 4,975,374to Goodman et al., disclose nucleotide sequences of glutamine synthetasegenes which confer resistance to herbicides such as L-phosphinothricin.The nucleotide sequence of a PAT gene is provided in Europeanapplication No. 0 242 246 to Leemans et al. DeGreef et al.,Bio/Technology 7:61 (1989) describe the production of transgenic plantsthat express chimeric bar genes coding for phosphinothricin acetyltransferase activity. Exemplary of genes conferring resistance tophenoxy proprionic acids and cyclohexones, such as sethoxydim andhaloxyfop are the Acc1-S1, Acc1-S2, and Acc2-S3 genes described byMarshall et al., Theor. Appl. Genet. 83:435 (1992).

C. An herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+genes) or a benzonitrile (nitrilase gene). Przibila et al., PlantCell 3:169 (1991), describe the transformation of Chlamydomonas withplasmids encoding mutant psbA genes. Nucleotide sequences for nitrilasegenes are disclosed in U.S. Pat. No. 4,810,648 to Stalker and DNAmolecules containing these genes are available under ATCC Accession Nos.53435, 67441 and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J.285:173 (1992).

D. Acetohydroxy acid synthase, which has been found to make plants thatexpress this enzyme resistant to multiple types of herbicides, has beenintroduced into a variety of plants. See Hattori et al., Mol. Gen.Genet. 246:419, 1995. Other genes that confer tolerance to herbicidesinclude a gene encoding a chimeric protein of rat cytochrome P4507A1 andyeast NADPH-cytochrome P450 oxidoreductase (Shiota et al., PlantPhysiol., 106:17, 1994), genes for glutathione reductase and superoxidedismutase (Aono et al., Plant Cell Physiol. 36:1687, 1995), and genesfor various phosphotransferases (Datta et al., Plant Mol. Biol. 20:619,1992).

E. Protoporphyrinogen oxidase (protox) is necessary for the productionof chlorophyll, which is necessary for all plant survival. The protoxenzyme serves as the target for a variety of herbicidal compounds. Theseherbicides also inhibit growth of all the different species of plantspresent, causing their total destruction. The development of plantscontaining altered protox activity which are resistant to theseherbicides are described in U.S. Pat. Nos. 6,288,306; 6,282,837;5,767,373; and international publication WO 01/12825.

3. Genes that Confer or Contribute to a Value-Added Trait, such as:

A. Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearyl-ACP desaturase to increase stearicacid content of the plant. See Knultzon et al., Proc. Natl. Acad. Sci.USA 89:2625 (1992).

B. Decreased phytate content—1) Introduction of a phytase-encoding genewould enhance breakdown of phytate, adding more free phosphate to thetransformed plant. For example, see Van Hartingsveldt et al., Gene127:87 (1993), for a disclosure of the nucleotide sequence of anAspergillus niger phytase gene. 2) A gene could be introduced thatreduced phytate content. In maize, for example, this could beaccomplished by cloning and then reintroducing DNA associated with thesingle allele which is responsible for maize mutants characterized bylow levels of phytic acid. See Raboy et al., Maydica 35:383 (1990).

C. Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bacteriol. 170:810(1988) (nucleotide sequence of Streptococcus mutantsfructosyltransferase gene), Steinmetz et al., Mol. Gen. Genet. 20:220(1985) (nucleotide sequence of Bacillus subtilis levansucrase gene), Penet al., Bio/Technology 10:292 (1992) (production of transgenic plantsthat express Bacillus licheniformis α-amylase), Elliot et al., PlantMolec. Biol. 21:515 (1993) (nucleotide sequences of tomato invertasegenes), S{acute over (ø)}gaard et al., J. Biol. Chem. 268:22480 (1993)(site-directed mutagenesis of barley α-amylase gene), and Fisher et al.,Plant Physiol. 102:1045 (1993) (maize endosperm starch branching enzymeII).

D. Elevated oleic acid via FAD-2 gene modification and/or decreasedlinolenic acid via FAD-3 gene modification. See U.S. Pat. Nos.6,063,947; 6,323,392; and international publication WO 93/11245.

4. Genes that Control Male Sterility

A. Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN—Ac—PPT. See international publication WO 01/29237.

B. Introduction of various stamen-specific promoters. See internationalpublications WO 92/13956 and WO 92/13957.

Introduction of the barnase and the barstar genes. See Paul et al.,Plant Mol. Biol. 19:611–622, 1992).

Methods for Soybean Transformation—Numerous methods for planttransformation have been developed including biological and physicalplant transformation protocols. See, for example, Miki et al.,“Procedures for Introducing Foreign DNA into Plants” in Methods in PlantMolecular Biology and Biotechnology, Glick, B. R. and Thompson, J. E.Eds. (CRC Press, Inc. Boca Raton, 1993) pages 67–88. In addition,expression vectors and in-vitro culture methods for plant cell or tissuetransformation and regeneration of plants are available. See, forexample, Gruber et al., “Vectors for Plant Transformation” in Methods inPlant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J.E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages 89–119.

A. Agrobacterium-mediated Transformation—One method for introducing anexpression vector into plants is based on the natural transformationsystem of Agrobacterium. See, for example, Horsch et al., Science227:1229 (1985). A. tumefaciens and A. rhizogenes are plant pathogenicsoil bacteria which genetically transform plant cells. The Ti and Riplasmids of A. tumefaciens and A. rhizogenes, respectively, carry genesresponsible for genetic transformation of the plant. See, for example,Kado, C. I., Crit. Rev. Plant Sci. 10:1 (1991). Descriptions ofAgrobacterium vector systems and methods for Agrobacterium-mediated genetransfer are provided by Gruber et al., supra, Miki et al., supra andMoloney et al., Plant Cell Reports 8:238 (1989). See also, U.S. Pat. No.5,563,055 (Townsend and Thomas), issued Oct. 8, 1996.

B. Direct Gene Transfer—Several methods of plant transformationcollectively referred to as direct gene transfer have been developed asan alternative to Agrobacterium-mediated transformation. A generallyapplicable method of plant surface of microprojectiles measuring 1 to 4μm. The expression vector is introduced into plant tissues with abiolistic device that accelerates the microprojectiles to speeds of 300to 600 m/s which is sufficient to penetrate plant cell walls andmembranes. Sanford et al., Part. Sci. Technol. 5:27 (1987); Sanford, J.C., Trends Biotech. 6:299 (1988); Klein et al., Bio/Tech. 6:559–563(1988); Sanford, J. C. Physiol Plant 7:206 (1990); Klein et al.,Biotechnology 10:268 (1992). See also U.S. Pat. No. 5,015,580 (Christou,et al.), issued May 14, 1991 and U.S. Pat. No. 5,322,783 (Tomes, etal.), issued Jun. 21, 1994.

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., Bio/Technology 9:996 (1991). Alternatively,liposome and spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4:2731 (1985); Christouet al., Proc Natl. Acad. Sci. USA 84:3962 (1987). Direct uptake of DNAinto protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-ornithine has also been reported. Hain et al., Mol. Gen. Genet.199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. Donn et al., In Abstracts of VIIth InternationalCongress on Plant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990);D'Halluin et al., Plant Cell 4:1495–1505 (1992) and Spencer et al.,Plant Mol. Biol. 24:51–61 (1994).

Following transformation of soybean target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic variety. The transgenic variety could then becrossed with another (non-transformed or transformed) variety in orderto produce a new transgenic variety. Alternatively, a genetic trait thathas been engineered into a particular soybean line using the foregoingtransformation techniques could be moved into another line usingtraditional backcrossing techniques that are well known in the plantbreeding arts. For example, a backcrossing approach could be used tomove an engineered trait from a public, non-elite variety into an elitevariety, or from a variety containing a foreign gene in its genome intoa variety or varieties that do not contain that gene. As used herein,“crossing” can refer to a simple X by Y cross or the process ofbackcrossing depending on the context.

Single-Gene Conversions—When the term soybean plant is used in thecontext of the present invention, this also includes any single geneconversions of that variety. The term single gene converted plant asused herein refers to those soybean plants which are developed by aplant breeding technique called backcrossing wherein essentially all ofthe desired morphological and physiological characteristics of a varietyare recovered in addition to the single gene transferred into thevariety via the backcrossing technique. Backcrossing methods can be usedwith the present invention to improve or introduce a characteristic intothe variety. The term backcrossing as used herein refers to the repeatedcrossing of a hybrid progeny back to the recurrent parent, i.e.,backcrossing 1, 2, 3, 4, 5, 6, 7, 8, 9 or more times to the recurrentparent. The parental soybean plant that contributes the gene for thedesired characteristic is termed the nonrecurrent or donor parent. Thisterminology refers to the fact that the nonrecurrent parent is used onetime in the backcross protocol and therefore does not recur. Theparental soybean plant to which the gene or genes from the nonrecurrentparent are transferred is known as the recurrent parent as it is usedfor several rounds in the backcrossing protocol (Poehlman & Sleper,1994; Fehr, 1987). In a typical backcross protocol, the original varietyof interest (recurrent parent) is crossed to a second variety(nonrecurrent parent) that carries the single gene of interest to betransferred. The resulting progeny from this cross are then crossedagain to the recurrent parent and the process is repeated until asoybean plant is obtained wherein essentially all of the desiredmorphological and physiological characteristics of the recurrent parentare recovered in the converted plant, in addition to the singletransferred gene from the nonrecurrent parent, as determined at the 5%significance level when grown in the same environmental conditions.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalvariety. To accomplish this, a single gene of the recurrent variety ismodified or substituted with the desired gene from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphological,constitution of the original variety. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross; one ofthe major purposes is to add some agronomically important trait to theplant. The exact backcrossing protocol will depend on the characteristicor trait being altered to determine an appropriate testing protocol.Although backcrossing methods are simplified when the characteristicbeing transferred is a dominant allele, a recessive allele may also betransferred. In this instance it may be necessary to introduce a test ofthe progeny to determine if the desired characteristic has beensuccessfully transferred.

Many single gene traits have been identified that are not regularlyselected for in the development of a new variety but that can beimproved by backcrossing techniques. Single gene traits may or may notbe transgenic; examples of these traits include but are not limited to,male sterility, waxy starch, herbicide resistance, resistance forbacterial, fungal, or viral disease, insect resistance, male fertility,enhanced nutritional quality, industrial usage, yield stability andyield enhancement. These genes are generally inherited through thenucleus. Several of these single gene traits are described in U.S. Pat.Nos. 5,959,185; 5,973,234 and 5,977,445; the disclosures of which arespecifically hereby incorporated by reference.

Further reproduction of the variety can occur by tissue culture andregeneration. Tissue culture of various tissues of soybeans andregeneration of plants therefrom is well known and widely published. Forexample, reference may be had to Komatsuda, T. et al., Crop Sci.31:333–337 (1991); Stephens, P. A., et al., Theor. Appl. Genet. (1991)82:633–635; Komatsuda, T. et al., Plant Cell, Tissue and Organ Culture,28:103–113 (1992); Dhir, S. et al., Plant Cell Reports (1992)11:285–289; Pandey, P. et al., Japan J. Breed. 42:1–5 (1992); andShetty, K., et al., Plant Science 81:245–251 (1992); as well as U.S.Pat. No. 5,024,944 issued Jun. 18, 1991 to Collins et al., and U.S. Pat.No. 5,008,200 issued Apr. 16, 1991 to Ranch et al. Thus, another aspectof this invention is to provide cells which upon growth anddifferentiation produce soybean plants having the physiological andmorphological characteristics of soybean variety 4442112.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, plant clumps, and plantcells that can generate tissue culture that are intact in plants orparts of plants, such as embryos, pollen, flowers, seeds, pods, leaves,stems, roots, root tips, anthers, and the like. Means for preparing andmaintaining plant tissue culture are well known in the art. By way ofexample, a tissue culture comprising organs has been used to produceregenerated plants. U.S. Pat. Nos. 5,959,185; 5,973,234 and 5,977,445describe certain techniques, the disclosures of which are incorporatedherein by reference.

This invention also is directed to methods for producing a soybean plantby crossing a first parent soybean plant with a second parent soybeanplant wherein the first or second parent soybean plant is a soybeanplant of the variety 4442112. Further, both first and second parentsoybean plants can come from the soybean variety 4442112. Thus, any suchmethods using the soybean variety 4442112 are part of this invention:selfing, backcrosses, hybrid production, crosses to populations, and thelike. All plants produced using soybean variety 4442112 as a parent arewithin the scope of this invention, including those developed fromvarieties derived from soybean variety 4442112. Advantageously, thesoybean variety could be used in crosses with other, different, soybeanplants to produce the first generation (F₁) soybean hybrid seeds andplants with superior characteristics. The variety of the invention canalso be used for transformation where exogenous genes are introduced andexpressed by the variety of the invention. Genetic variants createdeither through traditional breeding methods using variety 4442112 orthrough transformation of 4442112 by any of a number of protocols knownto those of skill in the art are intended to be within the scope of thisinvention.

The following describes breeding methods that may be used with cultivar4442112 in the development of further soybean plants. One suchembodiment is a method for developing a 4442112 progeny soybean plant ina soybean plant breeding program comprising: obtaining the soybeanplant, or a part thereof, of cultivar 4442112 utilizing said plant orplant part as a source of breeding material and selecting a 4442112progeny plant with molecular markers in common with 4442112 and/or withmorphological and/or physiological characteristics selected from thecharacteristics listed in Tables 1 or 2. Breeding steps that may be usedin the soybean plant breeding program include pedigree breeding, backcrossing, mutation breeding, and recurrent selection. In conjunctionwith these steps, techniques such as RFLP-enhanced selection, geneticmarker enhanced selection (for example SSR markers) and the making ofdouble haploids may be utilized.

Another method involves producing a population of cultivar 4442112progeny soybean plants, comprising crossing cultivar 4442112 withanother soybean plant, thereby producing a population of soybean plants,which, on average, derive 50% of their alleles from cultivar 4442112. Aplant of this population may be selected and repeatedly selfed or sibbedwith a soybean cultivar resulting from these successive filialgenerations. One embodiment of this invention is the soybean cultivarproduced by this method and that has obtained at least 50% of itsalleles from cultivar 4442112.

One of ordinary skill in the art of plant breeding would know how toevaluate the traits of two plant varieties to determine if there is nosignificant difference between the two traits expressed by thosevarieties. For example, see Fehr and Walt, Principles of CultivarDevelopment, p 261–286 (1987). Thus the invention includes soybeancultivar 4442112 progeny soybean plants comprising a combination of atleast two 4442112 traits selected from the group consisting of thoselisted in Tables 1 and 2 or the 4442112 combination of traits listed inthe Summary of the Invention, so that said progeny soybean plant is notsignificantly different for said traits than soybean cultivar 4442112 asdetermined at the 5% significance level when grown in the sameenvironment. Using techniques described herein, molecular markers may beused to identify said progeny plant as an 4442112 progeny plant. Meantrait values may be used to determine whether trait differences aresignificant, and preferably the traits are measured on plants grownunder the same environmental conditions. Once such a variety isdeveloped its value is substantial since it is important to advance thegermplasm base as a whole in order to maintain or improve traits such asyield, disease resistance, pest resistance, and plant performance inextreme environmental conditions.

Progeny of cultivar 4442112 may also be characterized through theirfilial relationship with soybean cultivar 4442112, as for example, beingwithin a certain number of breeding crosses of soybean cultivar 4442112.A breeding cross is a cross made to introduce new genetics into theprogeny, and is distinguished from a cross, such as a self or a sibcross, made to select among existing genetic alleles. The lower thenumber of breeding crosses in the pedigree, the closer the relationshipbetween soybean cultivar 4442112 and its progeny. For example, progenyproduced by the methods described herein may be within 1, 2, 3, 4 or 5breeding crosses of soybean cultivar 4442112.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which soybean plants can be regenerated,plant calli, plant clumps, and plant cells that are intact in plants orparts of plants, such as embryos, pollen, ovules, flowers, pods, leaves,roots, root tips, anthers, and the like.

INDUSTRIAL USES

The seed of soybean variety 4442112, the plant produced from the seed,the hybrid soybean plant produced from the crossing of the variety withany other soybean plant, hybrid seed, and various parts of the hybridsoybean plant can be utilized for human food, livestock feed, and as araw material in industry.

The soybean is the world's leading source of vegetable oil and proteinmeal. The oil extracted from soybeans is used for cooking oil,margarine, and salad dressings. Soybean oil is composed of saturated,monounsaturated and polyunsaturated fatty acids. It has a typicalcomposition of 11% palmitic, 4% stearic, 25% oleic, 50% linoleic and 9%linolenic fatty acid content (“Economic Implications of Modified SoybeanTraits Summary Report”, Iowa Soybean Promotion Board and AmericanSoybean Association Special Report 92S, May 1990). Changes in fatty acidcomposition for improved oxidative stability and nutrition areconstantly sought after. Industrial uses of soybean oil which issubjected to further processing include ingredients for paints,plastics, fibers, detergents, cosmetics, lubricants and biodiesel fuel.Soybean oil may be split, inter-esterified, sulfurized, epoxidized,polymerized, ethoxylated, or cleaved. Designing and producing soybeanoil derivatives with improved functionality and improved oliochemistryis a rapidly growing field. The typical mixture of triglycerides isusually split and separated into pure fatty acids, which are thencombined with petroleum-derived alcohols or acids, nitrogen, sulfonates,chlorine, or with fatty alcohols derived from fats and oils.

Soybean is also used as a food source for both animals and humans.Soybean is widely used as a source of protein for animal feeds forpoultry, swine and cattle. During processing of whole soybeans, thefibrous hull is removed and the oil is extracted. The remaining soybeanmeal is a combination of carbohydrates and approximately 50% protein.

For human consumption soybean meal is made into soybean flour which isprocessed to protein concentrates used for meat extenders or specialtypet foods. Production of edible protein ingredients from soybean offersa healthy, less expensive replacement for animal protein in meats aswell as dairy-type products.

Tables

In Table 2 that follows, the traits and characteristics of soybeancultivar 4442112 are compared to several competing varieties ofcommercial soybeans of similar maturity. In the tables, column 1 showsthe comparison number; column 2 is the year of the test; columns 3 and 4give the number of locations and number of observations, respectively.Column 5 indicates the genotype and column 6 shows the mean yield.Column 7 presents the t value and columns 8 and 9 present the critical tvalues at the 0.05% and 0.01% levels of significance, respectively.

As shown in Table 2, soybean cultivar 4442112 yields higher than 24commercial varieties with the increase over all comparisons beingsignificant at the 0.01 level of probability.

TABLE 2 PAIRED COMPARISONS Comp # of # of Mean Critical Critical # YearLoc. Obs. Genotype Yield t Value t @ .05 t @ .01 1 2004 7 20 444211245.0 8.61** 1.73 2.54 JG900213RJ 32.2 2 2004 7 18 4442112 45.9 8.83**1.74 2.57 CSR0030 29.7 3 2004 7 20 4442112 44.9 6.10** 1.73 2.54CSR00523 37.8 4 2004 7 20 4442112 45.3 6.17** 1.73 2.54 CSRX00504 36.3 52004 7 21 4442112 45.2 2.66** 1.72 2.53 JG900511RRJ 40.4 6 2004 7 214442112 45.2 8.54** 1.72 2.53 JG900613RJ 36.3 7 2004 7 18 4442112 44.59.83** 1.74 2.57 CSRX00704 33.5 8 2004 7 20 4442112 45.3 4.96** 1.732.54 AG00802 36.3 9 2004 7 21 4442112 45.2 5.23** 1.72 2.53 NKS00-J433.1 10 2004 7 20 4442112 45.0 6.67** 1.73 2.54 CSR0094-24 37.2 11 20047 21 4442112 45.2 3.28** 1.72 2.53 DKB009-51 40.7 12 2004 7 21 444211245.2 5.00** 1.72 2.53 JG900012RJ 38.8 13 2004 7 21 4442112 45.2 4.62**1.72 2.53 CSR0234 39.2 14 2004 7 20 4442112 45.2 3.49** 1.73 2.54 AG010139.2 15 2004 7 21 4442112 45.2 12.05** 1.72 2.53 BPR13255 34.1 16 2004 720 4442112 45.6 5.17** 1.73 2.54 CSR0324 38.6 17 2004 7 21 4442112 45.22.74** 1.72 2.53 CSR0220 42.7 18 2004 7 19 4442112 45.2 10.43** 1.732.55 CSR0254 31.5 19 2004 7 21 4442112 45.2 2.54** 1.72 2.53 CSR032042.2 20 2004 7 21 4442112 45.2 3.97** 1.72 2.53 CSR00934 39.8 21 2004 720 4442112 45.0 3.32** 1.73 2.54 FLS2602R 41.4 22 2004 7 21 4442112 45.23.80** 1.72 2.53 NKS02-G2 40.2 23 2004 7 21 4442112 45.2 3.71** 1.722.53 P90M20 39.8 24 2004 7 21 4442112 45.2 3.09** 1.72 2.53 AG0301 41.6**Significant at .01 level of probability *Significant at .05 level ofprobability

Deposit Information

A deposit of the Mertec LLC proprietary soybean cultivar designated4442112 disclosed above and recited in the appended claims has been madewith the American Type Culture Collection (ATCC), 10801 UniversityBoulevard, Manassas, Va. 20110. The date of deposit was Sep. 20, 2005.The deposit of 2,500 seeds was taken from the same deposit maintained byMertec LLC since prior to the filing date of this application. Allrestrictions upon the deposit have been removed, and the deposit isintended to meet all of the requirements of 37 C.F.R. 1.801–1.809. TheATCC accession number is PTA-6983. The deposit will be maintained in thedepository for a period of 30 years, or 5 years after the last request,or for the effective life of the patent, whichever is longer, and willbe replaced as necessary during that period.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding. However, it will be obvious that certain changes andmodifications such as single gene modifications and mutations,somoclonal variants, variant individuals selected from large populationsof the plants of the instant variety and the like may be practicedwithin the scope of the invention as limited only by the scope of theappended claims.

1. A seed of soybean cultivar 4442112, wherein a representative sampleof seed of said cultivar was deposited under ATCC Accession No.PTA-6983.
 2. A soybean plant, or a part thereof, produced by growing theseed of claim
 1. 3. A tissue culture of cells produced from the plant ofclaim 2, wherein said cells of the tissue culture are produced from aplant part selected from the group consisting of leaves, pollen,embryos, cotyledons, hypocotyls, meristematic cells, roots, root tips,pistils, anthers, flowers, stems and pods.
 4. A protoplast produced fromthe plant of claim 2 or the tissue culture of claim
 3. 5. A soybeanplant regenerated from the tissue culture of claim 3, wherein the planthas all the morphological and physiological characteristics of cultivar4442112.
 6. A method for producing an F1 hybrid soybean seed, whereinthe method comprises crossing the plant of claim 2 with a differentsoybean plant and harvesting the resultant F1 hybrid soybean seed.
 7. Amethod of producing an herbicide resistant soybean plant wherein themethod comprises transforming the soybean plant of claim 2 with atransgene wherein the transgene confers resistance to an herbicideselected from the group consisting of imidazolinone, sulfonylurea,glyphosate, glufosinate, L-phosphinothricin, triazine and benzonitrile.8. An herbicide resistant soybean plant produced by the method of claim7.
 9. A method of producing an insect resistant soybean plant whereinthe method comprises transforming the soybean plant of claim 2 with atransgene that confers insect resistance.
 10. An insect resistantsoybean plant produced by the method of claim
 9. 11. The soybean plantof claim 10, wherein the transgene encodes a Bacillus thuringiensisendotoxin.
 12. A method of producing a disease resistant soybean plantwherein the method comprises transforming the soybean plant of claim 2with a transgene that confers disease resistance.
 13. A diseaseresistant soybean plant produced by the method of claim
 12. 14. A methodof producing a soybean plant with modified fatty acid metabolism ormodified carbohydrate metabolism wherein the method comprisestransforming the soybean plant of claim 2 with a transgene encoding aprotein selected from the group consisting of phytase,fructosyltransferase, levansucrase, α-amylase, invertase and starchbranching enzyme or encoding the antisense of a stearyl-ACP desaturasegene, wherein the transgene is expressed and the fatty acid metabolismor the carbohydrate metabolism of the soybean plant is modified.
 15. Asoybean plant having modified fatty acid metabolism or modifiedcarbohydrate metabolism produced by the method of claim
 14. 16. A methodof introducing a desired trait into soybean cultivar 4442112 wherein themethod comprises: (a) crossing a 4442112 plant, wherein a representativesample of seed was deposited under ATCC Accession No. PTA-6983, with aplant of another soybean cultivar that comprises a desired trait toproduce progeny plants wherein the desired trait is selected from thegroup consisting of male sterility, herbicide resistance, insectresistance, modified fatty acid metabolism, modified carbohydratemetabolism and resistance to bacterial disease, fungal disease or viraldisease; (b) selecting one or more progeny plants that have the desiredtrait to produce selected progeny plants; (c) crossing the selectedprogeny plants with the 4442112 plants to produce backcross progenyplants; (d) selecting for backcross progeny plants that have the desiredtrait and the physiological and morphological characteristics of soybeancultivar 4442112 listed in Table 1 to produce selected backcross progenyplants; and (e) repeating steps (c) and (d) three or more times insuccession to produce selected fourth or higher backcross progeny plantsthat comprise the desired trait and all of the physiological andmorphological characteristics of soybean cultivar 4442112 listed inTable
 1. 17. A plant produced by the method of claim 16 wherein theplant has the desired trait and all of the physiological andmorphological characteristics of soybean cultivar 4442112 listed inTable
 1. 18. The plant of claim 17, wherein the desired trait isherbicide resistance and the resistance is conferred to an herbicideselected from the group consisting of imidazolinone, sulfonylurea,glyphosate, glufosinate, L-phosphinothricin, triazine and benzonitrile.19. The plant of claim 17, wherein the desired trait is insectresistance and the insect resistance is conferred by a transgeneencoding a Bacillus thuringiensis endotoxin.
 20. The plant of claim 17,wherein the desired trait is modified fatty acid metabolism or modifiedcarbohydrate metabolism and said desired trait is conferred by a nucleicacid encoding a protein selected from the group consisting of phytase,fructosyltransferase, levansucrase, α-amylase, invertase and starchbranching enzyme or encoding an antisense of stearyl-ACP desaturase.